|Budget Amount *help
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
The aim of this study is to find out a primary factor that regulates a flow of de novo pyrimidine biosynthetic pathway in Toxoplasma gondii within term of the project. To perform a study of the project, the cellular contents of nucleic acid precursors, purine and pyrimidine nucleotides, were investigated by examining an analytical method to measure the compounds. The nucleotide concentrations in T.gondii cell were measurable by a high performance liquid chromatography and the sensitivity of the measurement was within an experimental condition. For another approach to the project, a cloning of the gene encoding carbamylphosphate synthetase-II (CPS-II) that is the first step enzyme and the most important enzyme of pyrimidine de novo biosynthetic pathway, was attempted. Consequently, a partial cDNA clone encoding a substrate binding site of CPS-II was obtained, however, full length of CPS-II gene had been reported by a group of Dartmouth University in the United States before our success.
Anyway, it was clearly identified that there is no PRPP, the most potent activator of CPS-II, binding site in the T.gondii CPS-II from our results and the data from the group of Dartmouth University. We started a collaboration project with the group of Dartmouth University to study an enzyme regulation of T.gondii CPS-II and tried to make a full size of recombinant CPS-II. Unfortunately, the expression of the recombinant CPS-II has not been successful because T.gondii CPS-II is a huge protein. Although an enzymological study has not been performed, the data of full amino acid sequence of CPS-II strongly suggests that the most important factor that contributes to the flow of de novo pyrimidine biosynthetic pathway is a supply of ATP, a substrate of CPS-II. An accurate assay of ATP concentration in T.gondii cell is very hard because of the ATP contamination from host cell, nevertheless, the estimated concentration of ATP in growing tachyzoite cell (〜5 mM) is quite higher than that of dormant cell (〜1 mM). Considering the km value of T.gondii CPS-II for ATP (〜10 mM), almost no enzyme activity is probably present under dormant condition. The results strongly suggest that the supply of energy is the most important factor for the flow of de novo pyrimidine biosynthesis. The aim of this project was almost achieved, though many facts should be further confirmed. Less